Cysteine cathepsins and extracellular matrix degradation

Background

Cysteine cathepsins are normally found in the lysosomes where they are involved in intracellular protein turnover. Their ability to degrade the components of the extracellular matrix in vitro was first reported more than 25 years ago. However, cathepsins were for a long time not considered to be among the major players in ECM degradation in vivo. During the last decade it has, however, become evident that abundant secretion of cysteine cathepsins into extracellular milieu is accompanying numerous physiological and disease conditions, enabling the cathepsins to degrade extracellular proteins.

Scope of view

In this review we will focus on cysteine cathepsins and their extracellular functions linked with ECM degradation, including regulation of their activity, which is often enhanced by acidification of the extracellular microenvironment, such as found in the bone resorption lacunae or tumor microenvironment. We will further discuss the ECM substrates of cathepsins with a focus on collagen and elastin, including the importance of that for pathologies. Finally, we will overview the current status of cathepsin inhibitors in clinical development for treatment of ECM-linked diseases, in particular osteoporosis.

Major conclusions

Cysteine cathepsins are among the major proteases involved in ECM remodeling, and their role is not limited to degradation only. Deregulation of their activity is linked with numerous ECM-linked diseases and they are now validated targets in a number of them. Cathepsins S and K are the most attractive targets, especially cathepsin K as a major therapeutic target for osteoporosis with drugs targeting it in advanced clinical trials.

General significance

Due to their major role in ECM remodeling cysteine cathepsins have emerged as an important group of therapeutic targets for a number of ECM-related diseases, including, osteoporosis, cancer and cardiovascular diseases. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

The cell surface: the stage for matrix metalloproteinase regulation of migration

Matrix metalloproteinases are important for the turnover of extracellular matrix in tissue. Recent studies have expanded their roles well beyond extracellular matrix degradation - they also cleave many growth factors, cytokines and cell adhesion molecules in the extracellular milieu, modulating their functions irreversibly. In particular, some matrix metalloproteinases that associate with the cell surface have arisen as intriguing regulators of cellular functions, including migration.     

Cathepsin regulation on microglial function

Microglia, the resident mononuclear phagocyte population in the brain, have long been implicated in the pathology of neurodegenerative age-associated disorders. However, activated microglia have now been identified as homeostatic keepers in the brain, because they are involved in the initiation and resolution of neuropathology. The complex roles of activated microglia appear to be linked to change from inflammatory and neurotoxic to anti-inflammatory and neuroprotective phenotypes. Increased expression and secretion of various cathepsins support roles of activated microglia in chronic neuroinflammation, the neurotoxic M1-like polarization and neuronal death. Moreover, changes in expression and localization of microglial cathepsin B play a critical role in the acceleration of the brain aging. Beyond the role as brain-resident macrophages, many lines of evidence have shown that microglia have essential roles in the maturation and maintenance of neuronal circuits in the developing and adult brain. Cathepsin S secreted from microglia induces the diurnal variation of spine density of cortical neurons though proteolytic modification of peri-synaptic extracellular matrix molecules. In this review, I highlight the emerging roles of cathepsins that support the roles of microglia in both normal healthy and pathological brains. In addition, I discuss cathepsin inhibitors as potential therapeutic targets for brain disorders.     

Cysteinyl cathepsins in cardiovascular diseases

Cysteinyl cathepsins are lysosomal/endosomal proteases that mediate bulk protein degradation in these intracellular acidic compartments. Yet, studies indicate that these proteases also appear in the nucleus, nuclear membrane, cytosol, plasma membrane, and extracellular space. Patients with cardiovascular diseases (CVD) show increased levels of cathepsins in the heart, aorta, and plasma. Plasma cathepsins often serve as biomarkers or risk factors of CVD. In aortic diseases, such as atherosclerosis and abdominal aneurysms, cathepsins play pathogenic roles, but many of the same cathepsins are cardioprotective in hypertensive, hypertrophic, and infarcted hearts. During the development of CVD, cathepsins are regulated by inflammatory cytokines, growth factors, hypertensive stimuli, oxidative stress, and many others. Cathepsin activities in inflammatory molecule activation, immunity, cell migration, cholesterol metabolism, neovascularization, cell death, cell signaling, and tissue fibrosis all contribute to CVD and are reviewed in this article in memory of Dr. Nobuhiko Katunuma for his contribution to the field.     

Lysosomal pathways to cell death and their therapeutic applications

Lysosomes are the major cell digestive organelles that were discovered over 50 years ago. They contain a number of hydrolases that help them to degrade intracellular and extracellular material delivered. Among the hydrolases, the cathepsins, a group of proteases enclosed in the lysosomes, have a major role. About a decade ago, the cathepsins were found to participate in apoptosis. Following their release into the cytosol, they cleave Bid and degrade antiapoptotic Bcl-2 proteins, thereby triggering the mitochondrial pathway of apoptosis, with the lysosomal membrane permeabilization being the critical step in this pathway. Lysosomal dysfunction is linked with several diseases, including cancer and neurodegenerative disorders, thereby providing a potential for therapeutic applications. In this review lysosomes and lysosomal proteases involvement in apoptosis and their possible pharmaceutical targeting are discussed.     

Cysteine cathepsins in human cancer

Proteases play causal roles in the malignant progression of human tumors. This review centers on the roles in this process of cysteine cathepsins, i.e., peptidases belonging to the papain family (C1) of the CA clan of cysteine proteases. Cysteine cathepsins, most likely along with matrix metalloproteases (MMPs) and serine proteases, degrade the extracellular matrix, thereby facilitating growth and invasion into surrounding tissue and vasculature. Studies on tumor tissues and cell lines have shown changes in expression, activity and distribution of cysteine cathepsins in numerous human cancers. Molecular, immunologic and pharmacological strategies to modulate expression and activity of cysteine cathepsins have provided evidence for a causal role for these enzymes in tumor progression and invasion. Clinically, the levels, activities and localization of cysteine cathepsins and their endogenous inhibitors have been shown to be of diagnostic and prognostic value. Understanding the roles that cysteine proteases play in cancer could lead to the development of more efficacious therapies.     

Lung cysteine cathepsins: intruders or unorthodox contributors to the kallikrein-kinin system?

The protease/antiprotease balance is tipped in favor of enhanced proteolysis in inflammatory lung disorders, promoting the spread and severity of inflammation. Cysteine cathepsins participate in the remodeling and/or degradation of the pulmonary extra cellular matrix and in lung homeostasis. There is now good evidence that cathepsins are involved in fibrosis, emphysema, asthma, and in bronchopulmonary dysplasia. Kinins are inflammatory mediators that induce edema, pain and vasodilatation, and participate in vascular homeostasis. Kinins may also contribute to the immune system by acting as danger signals, and activating bradykinin receptors. Kinins are believed to play a role in inflammatory obstructive airway diseases, asthma, and allergic rhinitis. Their release by plasma and tissue kallikreins is severely reduced at inflammatory sites, although local kinin production seems to remain intact. Such conflicting observations suggest that there are alternative mechanisms of kinin metabolism besides the classical pathways. This article reviews the biological and pathophysiological roles of lung cysteine cathepsins, kinins and their receptors, and summarizes the indications that cysteine cathepsins may contribute to kinin liberation and/or degradation.     

Cathepsins D and L reduce the toxicity of advanced glycation end products

Advanced glycation end product-modified proteins are known for accumulating during aging and in several pathological conditions such as diabetes, renal failure, and neurodegenerative disorders. There is little information about the intracellular fate of endocytosed advanced glycation end products (AGEs) and their influence on proteolytic systems. However, it is known that the lysosomal system is impaired during aging. Therefore, undegraded material may accumulate and play a considerable role in the development of diverse diseases. To investigate if AGEs can be degraded and to test whether they accumulate because of impaired lysosomal proteases we studied the effects of advanced glycation end products on the endosomal-lysosomal system. Five different types of AGEs were generated by bovine serum albumin incubation with glyoxal, methylglyoxal, glucose, fructose, and ribose. The first experiments revealed the uptake of AGEs by the macrophage cell line RAW 264.7. Further investigations demonstrated an increase in cathepsin D and L activity and an increase in mature cathepsins D and L. Increased activities were accompanied by the presence of more lysosomes, measured by staining with LysoTracker blue. To specify the roles of cathepsins D and L we used knockout cells to test the roles of both cathepsins on the toxicity of advanced glycation end products. In summary we conclude that both cathepsins are required for a reduction in advanced glycation end product-induced cytotoxicity.     

Transforming growth factor (TGF)-beta1 stimulates pulmonary fibrosis and inflammation via a Bax-dependent, bid-activated pathway that involves matrix metalloproteinase-12

Fibrosis, apoptosis, and the exaggerated production of transforming growth factor (TGF)-beta(1) are juxtaposed in a variety of pulmonary diseases including the interstitial lung diseases and asthma. In these disorders, the relationships between these responses are not well defined. In addition, the apoptosis pathways that contribute to these responses and the mechanism(s) of their contribution have not been described. We hypothesized that BH3 domain-only protein-induced apoptosis plays an important role in the pathogenesis of TGF-beta(1)-induced pulmonary responses. To test this hypothesis, we characterized the effects of transgenic TGF-beta(1) in mice with wild type (WT) and null Bax loci. To investigate the mechanisms of Bax activation and its effector functions, we also compared the effects of TGF-beta(1) in mice with WT and null Bid and matrix metalloproteinase (MMP)-12 loci, respectively. These studies demonstrate that TGF-beta(1) is a potent stimulator of Bax, Bid, and MMP-12. The studies also demonstrate that Bax and Bid play key roles in the pathogenesis of TGF-beta(1)-induced inflammation, fibrosis, and apoptosis; that TGF-beta(1) stimulates MMP-12, TIMP-1, and cathepsins and inhibits MMP-9 and p21 via Bax- and Bid-dependent mechanisms; and that TGF-beta(1)-stimulated pulmonary fibrosis is ameliorated in MMP-12-deficient animals. Finally, they demonstrate that Bax, Bid, and MMP-12 play similar roles in bleomycin-induced fibrosis, thereby highlighting the importance of this Bid-activated, Bax-mediated pathway and downstream MMP-12 in a variety of fibrogenic settings.     

Roles of matrix metalloproteinases in cancer progression and their pharmacological targeting

Matrix metalloproteinases (MMPs) consist of a multigene family of zinc-dependent extracellular matrix (ECM) remodeling endopeptidases implicated in pathological processes, such as carcinogenesis. In this regard, their activity plays a pivotal role in tumor growth and the multistep processes of invasion and metastasis, including proteolytic degradation of ECM, alteration of the cell-cell and cell-ECM interactions, migration and angiogenesis. The underlying premise of the current minireview is that MMPs are able to proteolytically process substrates in the extracellular milieu and, in so doing, promote tumor progression. However, certain members of the MMP family exert contradicting roles at different stages during cancer progression, depending among other factors on the tumor stage, tumor site, enzyme localization and substrate profile. MMPs are therefore amenable to therapeutic intervention by synthetic and natural inhibitors, providing perspectives for future studies. Multiple therapeutic agents, called matrix metalloproteinase inhibitors (MMPIs) have been developed to target MMPs, attempting to control their enzymatic activity. Even though clinical trials with these compounds do not show the expected results in most cases, the field of MMPIs is ongoing. This minireview critically evaluates the role of MMPs in relation to cancer progression, and highlights the challenges, as well as future prospects, for the design, development and efficacy of MMPIs.     

Importance of lysosomal cysteine proteases in lung disease

The human lysosomal cysteine proteases are a family of 11 proteases whose members include cathepsins B, C, H, L, and S. The biology of these proteases was largely ignored for decades because of their lysosomal location and the belief that their function was limited to the terminal degradation of proteins. In the past 10 years, this view has changed as these proteases have been found to have specific functions within cells. This review highlights some of these functions, specifically their roles in matrix remodeling and in regulating the immune response, and their relationship to lung diseases.     

The biochemical, biological, and pathological kaleidoscope of cell surface substrates processed by matrix metalloproteinases

Matrix metalloproteinases (MMPs) constitute a family of more than 20 endopeptidases. Identification of specific matrix and non-matrix components as MMP substrates showed that, aside from their initial role as extracellular matrix modifiers, MMPs play significant roles in highly complex processes such as the regulation of cell behavior, cell-cell communication, and tumor progression. Thanks to the comprehensive examination of the expanded MMP action radius, the initial view of proteases acting in the soluble phase has evolved into a kaleidoscope of proteolytic reactions connected to the cell surface. Important classes of cell surface molecules include adhesion molecules, mediators of apoptosis, receptors, chemokines, cytokines, growth factors, proteases, intercellular junction proteins, and structural molecules. Proteolysis of cell surface proteins by MMPs may have extremely diverse biological implications, ranging from maturation and activation, to inactivation or degradation of substrates. In this way, modification of membrane-associated proteins by MMPs is crucial for communication between cells and the extracellular milieu, and determines cell fate and the integrity of tissues. Hence, insights into the processing of cell surface proteins by MMPs and the concomitant effects on physiological processes as well as on disease onset and evolution, leads the way to innovative therapeutic approaches for cancer, as well as degenerative and inflammatory diseases.     

Cysteine Cathepsins in Neurological Disorders

Increased proteolytic activity is a hallmark of several pathological processes, including neurodegeneration. Increased expression and activity of cathepsins, lysosomal cysteine proteases, during degeneration of the central nervous system is frequently reported. Recent studies reveal that a disturbed balance of their enzymatic activities is the first insult in brain aging and age-related diseases. Leakage of cathepsins from lysosomes, due to their membrane permeability, and activation of pro-apoptotic factors additionally contribute to neurodegeneration. Furthermore, in inflammation-induced neurodegeneration the cathepsins expressed in activated microglia play a pivotal role in neuronal death. The proteolytic activity of cysteine cathepsins is controlled by endogenous protein inhibitors—the cystatins—which evidently fail to perform their function in neurodegenerative processes. Exogenous synthetic inhibitors, which may augment their inhibitory potential, are considered as possible therapeutic tools for the treatment of neurological disorders.     

Cysteine cathepsin non-inhibitory binding partners: modulating intracellular trafficking and function

Cysteine cathepsins play a fundamental role in tumor growth, invasion and migration, angiogenesis, and the metastatic cascade. Evidence of their overexpression in a wide array of human tumors has been well documented. Cysteine cathepsins seem to have a characteristic location-function relationship that leads to non-traditional roles such as those in development and pathology. For example, during tumor development, some cysteine cathepsins are found not just within lysosomes, but are also redistributed into presumptive exocytic vesicles at the cell periphery, resulting in their secretion. This altered localization contributes to non-lysosomal functions that have been linked to malignant progression. Mechanisms for altered localization are not well understood, but do include the interaction of cysteine cathepsins with binding partners that modulate intracellular trafficking and association with specific regions on the cell surface.     

Cysteine cathepsins: from structure, function and regulation to new frontiers

It is more than 50 years since the lysosome was discovered. Since then its hydrolytic machinery, including proteases and other hydrolases, has been fairly well identified and characterized. Among these are the cysteine cathepsins, members of the family of papain-like cysteine proteases. They have unique reactive-site properties and an uneven tissue-specific expression pattern. In living organisms their activity is a delicate balance of expression, targeting, zymogen activation, inhibition by protein inhibitors and degradation. The specificity of their substrate binding sites, small-molecule inhibitor repertoire and crystal structures are providing new tools for research and development. Their unique reactive-site properties have made it possible to confine the targets simply by the use of appropriate reactive groups. The epoxysuccinyls still dominate the field, but now nitriles seem to be the most appropriate "warhead". The view of cysteine cathepsins as lysosomal proteases is changing as there is now clear evidence of their localization in other cellular compartments. Besides being involved in protein turnover, they build an important part of the endosomal antigen presentation. Together with the growing number of non-endosomal roles of cysteine cathepsins is growing also the knowledge of their involvement in diseases such as cancer and rheumatoid arthritis, among others. Finally, cysteine cathepsins are important regulators and signaling molecules of an unimaginable number of biological processes. The current challenge is to identify their endogenous substrates, in order to gain an insight into the mechanisms of substrate degradation and processing. In this review, some of the remarkable advances that have taken place in the past decade are presented. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Human umbilical cord mesenchymal stem cell-derived extracellular vesicles: A novel therapeutic paradigm

Mesenchymal stem cells (MSCs) have been revealed to hold great potential for the development of new treatment approaches for various diseases. However, the clinical use of these cells is limited due to their tumorigenic effects. The therapeutic benefits of MSCs are largely dependent on paracrine factors including extracellular vesicles (EVs). EVs are nano-sized bilayer membrane structures containing lipids, microRNAs and proteins which play key roles in cell-to-cell communications. Because of their lower immunogenicity, tumorigenicity, and easier management, EVs have emerged as a new promising alternative to whole-cell therapy. Therefore, this paper reviews current preclinical studies on the use of EVs derived from human umbilical cord MSCs (hucMSCs) as a therapeutic approach in treatment of several diseases including neurological, cardiovascular, liver, kidney, and bone diseases as well as the cutaneous wound, inflammatory bowel disease, cancers, infertility, and other disorders.     

Minor proteins and enzymes of the Drosophila eggshell matrix

The Drosophila eggshell provides an in vivo model system for extracellular matrix assembly, in which programmed gene expression, cell migrations, extracellular protein trafficking, proteolytic processing, and cross-linking are all required to generate a multi-layered and regionally complex architecture. While abundant structural components of the eggshell are known and are being characterized, less is known about non-abundant structural, regulatory, and enzymatic components that are likely to play critical roles in eggshell assembly. We have used sensitive mass spectrometry-based analyses of fractionated eggshell matrices to validate six previously predicted eggshell proteins and to identify eleven novel components, and have characterized the expression patterns of many of their mRNAs. Among these are several putative structural or regulatory (non-enzymatic) proteins, most larger in mass than the major eggshell proteins and often showing preferential expression in follicle cells overlying specific structural features of the eggshell. Of particular note are the putative enzymes, some likely to be involved in matrix cross-linking (two yellow family members previously implicated in eggshell integrity, a heme peroxidase, and a small-molecule oxidoreductase) and others possibly involved in matrix proteolysis or adhesion (proteins related to cathepsins B and D). This work provides a framework for future molecular studies of eggshell assembly.     

Granzyme B in skin inflammation and disease

Granzyme B (GzmB) is a serine protease emerging as an important mediator of skin injury, inflammation and repair. Found at low levels in healthy skin, GzmB is dramatically elevated in chronic disease and inflammatory skin disorders, including diabetic ulcers, hypertrophic scarring, autoimmune skin disorders, cutaneous leishmaniasis and aging skin. Traditionally known for its pro-apoptotic function, the role of GzmB in disease has been redefined due to the discovery of additional activities involving the cleavage of extracellular matrix proteins, epithelial barrier disruption, fibrosis, vascular permeability, anoikis, inflammation and autoimmunity. In addition to the accumulation of GzmB⁺ cells in diseased tissue, and critical to the mechanistic redefinition, is the realization that GzmB often accumulates in the extracellular milieu, retains its activity in plasma, and is expressed by both immune and non-immune cells that may or may not express perforin, the pore-forming protein required for GzmB internalization into target cells. As GzmB is not normally found in the extracellular milieu, and does not appear to be regulated, GzmB-mediated proteolysis can impact processes such as tissue remodelling, barrier function, autoantigen generation and angiogenesis. The present review will summarize and critically examine the current knowledge regarding GzmB in inflammatory skin disease, providing an overview of both apoptotic and extracellular mechanisms, but with a focus on the extracellular roles of GzmB in skin health and disease.